Background:The permeability and physiological role of several large pore (hemi)channels are unresolved. Results: Large pore (hemi)channels, when heterologously expressed, display isoform-specific permeability and gating for ions and fluorescent dyes. Conclusion: Large pore channels have isoform-specific transport characteristics that can be used for their identification. Significance: Although large pore channels have characteristic properties in overexpression systems, these properties may be undetectable in native cells.

BackgroundType 2 diabetes is associated with abnormal electrical conduction and sudden cardiac death, but the pathogenic mechanism remains unknown. This study describes electrophysiological alterations in a diet-induced pre-diabetic rat model and examines the underlying mechanism.MethodsSprague–Dawley rats were fed either high-fat diet and fructose water or normal chow and water for 6 weeks. The electrophysiological properties of the whole heart was analyzed by in vivo surface ECG recordings, as wells as ex vivo in Langendorff perfused hearts during baseline, ischemia and re-perfussion. Conduction velocity was examined in isolated tissue strips. Ion channel and gap junction conductances were analyzed by patch-clamp studies in isolated cardiomyocytes. Fibrosis was examined by Masson’s Trichrome staining and thin-layer chromatography was used to analyze cardiac lipid content. Connexin43 (Cx43) expression and distribution was examined by western blotting and immunofluorescence respectively.ResultsFollowing 6 weeks of feeding, fructose-fat fed rats (FFFRs) showed QRS prolongation compared to controls (16.1 ± 0.51 (n = 6) vs. 14.7 ± 0.32 ms (n = 4), p < 0.05). Conduction velocity was slowed in FFFRs vs. controls (0.62 ± 0.02 (n = 13) vs. 0.79 ± 0.06 m/s (n = 11), p < 0.05) and Langendorff perfused FFFR hearts were more prone to ventricular fibrillation during reperfusion following ischemia (p < 0.05). The patch-clamp studies revealed no changes in Na+ or K+ currents, cell capacitance or gap junctional coupling. Cx43 expression was also unaltered in FFFRs, but immunofluorescence demonstrated an increased fraction of Cx43 localized at the intercalated discs in FFFRs compared to controls (78 ± 3.3 (n = 5) vs. 60 ± 4.2 % (n = 6), p < 0.01). No fibrosis was detected but FFFRs showed a significant increase in cardiac triglyceride content (1.93 ± 0.19 (n = 12) vs. 0.77 ± 0.13 nmol/mg (n = 12), p < 0.0001).ConclusionSix weeks on a high fructose-fat diet cause electrophysiological changes, which leads to QRS prolongation, decreased conduction velocity and increased arrhythmogenesis during reperfusion. These alterations are not explained by altered gap junctional coupling, Na+, or K+ currents, differences in cell size or fibrosis.

G α q -stimulation reduces intercellular coupling within 10 min via a decrease in the membrane lipid phosphatidylinositol-4,5-bisphosphate (PIP2), but the mechanism is unknown. Here we show that uncoupling in rat cardiomyocytes after stimulation of α -adrenergic G α q -coupled receptors with norepinephrine is prevented by proteasomal and lysosomal inhibitors, suggesting that internalization and possibly degradation of connexin43 (Cx43) is involved. Uncoupling was accompanied by increased Triton X-100 solubility of Cx43, which is considered a measure of the non-junctional pool of Cx43. However, inhibition of the proteasome and lysosome further increased solubility while preserving coupling, suggesting that communicating gap junctions can be part of the soluble fraction. Ubiquitination of Cx43 was also increased, and Cx43 co-immunoprecipitated with the ubiquitin ligase Nedd4. Conclusions : Norepinephrine increases ubiquitination of Cx43 in cardiomyocytes, possibly via Nedd4. We suggest that Cx43 is subsequently internalized, which is preceded by acquired solubility in Triton X-100, which does not lead to uncoupling per se .